A method for predicting individual residue contributions to enzyme specificity and binding-site energies, and its application to MTH1

Abstract

A new method for predicting the energy contributions to substrate binding and to specificity has been developed. Conventional global optimization methods do not permit the subtle effects responsible for these properties to be modeled with sufficient precision to allow confidence to be placed in the results, but by making simple alterations to the model, the precisions of the various energies involved can be improved from about ±2 kcal mol^-1 to ±0.1 kcal mol^-1. This technique was applied to the oxidized nucleotide pyrophosphohydrolase enzyme MTH1. MTH1 is unusual in that the binding and reaction sites are well separated-an advantage from a computational chemistry perspective, as it allows the energetics involved in docking to be modeled without the need to consider any issues relating to reaction mechanisms. In this study, two types of energy terms were investigated: the noncovalent interactions between the binding site and the substrate, and those responsible for discriminating between the oxidized nucleotide 8-oxo-dGTP and the normal dGTP. Both of these were investigated using the semiempirical method PM7 in the program MOPAC. The contributions of the individual residues to both the binding energy and the specificity of MTH1 were calculated by simulating the effect of mutations. Where comparisons were possible, all calculated results were in agreement with experimental observations. This technique provides fresh insight into the binding mechanism that enzymes use for discriminating between possible substrates.

Keywords: Binding; Docking; Enzyme specificity; MTH1; Noncovalent interactions; Nucleotide hydrolysis; PM7.

Fig. 1

MTH1 plus 8-oxo-dGMP substrate, showing the substrate in the binding site between two α-helices and in front of a β-sheet. In the binding site, the substrate forms five hydrogen bonds with the enzyme: one from Asp119, two from Asp120, and two from Asn33. Two water molecules, 2024 and 2134, also form hydrogen bonds with the substrate

Fig. 2

Atom numbering system for 8-oxo-guanine

Fig. 3

The D119–D120 anion in MTH1. The position of the ionizable hydrogen atom suggests that Asp119 exists as the carboxylate anion and that Asp120 exists as the neutral carboxylic acid

Fig. 4

Stereo view of residues in the binding pocket that do not form hydrogen bonds with the 8-oxo-dGMP substrate

Fig. 5

Hydrogen-bonding structure in the D119–D120–guanine complex

Fig. 6

Mutation D119A in MTH1 + 8-oxo-dGMP. In the D119A mutation, Asp120 spontaneously ionizes to form the carboxylate, which hydrogen bonds to neutral guanine

Fig. 7

Mutation D120A in MTH1 + 8-oxo-dGMP. In the D120A mutation, the position of the ionizable hydrogen atom suggests that Asp119 remains a neutral carboxylic acid which forms a strong hydrogen bond with the guanine anion

Fig. 8

Comparison of the Asp119–Asp120–guanine X-ray and PM7 environments. Left panel: structure from chain B in 3ZR0. Right panel: structure predicted using PM7